Technical Abstract:
Production of embryos in vitro has enormous potential for research and commercial applications in livestock. Unfortunately, in vitro production (IVP) of porcine embryos is extremely inefficient with these embryos developing inferior to their in vivo-produced (IVO) counterparts. Characterization of transcriptome profiles between IVO and IVP embryos would provide insight on potential biological processes that are compromised during IVP; thereby allowing scientists to target specific biological processes for the improvement of IVP methods. The objective of the current study was to compare global gene expression patterns from IVO and IVP embryos using SAR-SAGE. Whole-cell RNA from pools of day 6 IVO and IVP blastocysts served as template to generate amplified, sense RNA that was used to construct SAR-SAGE libraries. Sequence analysis of the IVO and IVP libraries yielded a total of 98,771 and 98,408 tags, respectively. A total of 20,029 and 23,453 unique putative transcripts were detected in the IVO and IVP libraries, respectively. Statistical analysis of SAGE tag frequencies between the IVO and IVP libraries indicated that 938 and 193 tags were differentially expressed at the P < 0.05 and P < 0.001 level of significance, respectively. Tentative annotation of the differentially expressed SAGE tags was determined using BLAST sequence alignment with the TIGR porcine specific gene index and NCBI human UniGene index. Annotated tags were caterogized into functional groupings according to gene ontology annotations. Real-time PCR was used to confirm differential expression for several transcripts from IVO and IVP blastocysts. These results demonstrate compromised gene expression from IVP blastocysts compared with IVO blastocysts for a number of physiological processes including cellular metabolism, transport and organization as well as response to stress.